The control of bleeding and clotting are extremely important in medical and surgical procedures. Protein C, a vitamin K-dependent plasma protein, is of major physiological importance in the control of bleeding. Protein C exists in two major forms, i.e., an inactive or nascent form and an active form. Active protein C ("APC") is a natural anticoagulant formed upon the proteolytic activation of protein C. This proteolytic activation is catalyzed in the blood by a thrombomodulin-thrombin complex.
Thrombin is an enzyme that has significant procoagulant activity. For example, it cleaves fibrinogen to form soluble fibrin, it activates platelets, and it converts the clotting cofactors V and VIII to their activated forms Va and VIIIa. Although it can activate protein C to form the natural anticoagulant APC, this activation is inhibited by physiological Ca.sup.2+ concentrations. Thrombin, however, can be inactivated by antithrombin (AT III).
Thrombomodulin (TM) is a 105 kD anionic transmembrane glycoprotein expressed on the surface of endothelial cells. Thrombomodulin strongly binds [K.sub.d 0.5 nM] the circulating enzyme thrombin (THR) and thereby dramatically alters the functional properties of thrombin. The complexation of THR by TM alters its substrate specificity so that the fibrinogen-cleaving activity of THR and the activation of platelets are curbed, i.e., attenuated. Also, the inactivation of thrombin by antithrombin is enhanced by this complexation. Furthermore, the altered substrate specificity of THR results in a significant acceleration of its proteolytic activation of circulating protein C to activated protein C, particularly in the presence of physiological Ca.sup.2+. APC, in turn, is a powerful anticoagulant serine protease which, in conjunction with protein S, terminates the procoagulant activity of Factors Va and VIIIa. See, C. T. Esmon, J. Biol. Chem., 264, 4743-4746 (1989), and W. A. Dittman et al., Blood, 75, 329-336 (1990). Thus, TM-dependent APC generation is crucial to the regulation of clotting. This is shown by the fulminant lethal thrombosis occurring in newborns with severe homozygous deficiencies of protein C, and the pronounced venous and arterial clotting diathesis that plagues some individuals heterozygous for protein C deficiency. See, U. Seligsohn et al., N. Engl. J. Med., 310, 559 (1984), and J. H. Griffin et al., J. Clin. Invest., 68, 1370 (1981).
In sum, the procoagulant activities of thrombin are inhibited by thrombomodulin (TM) in three distinct ways. It promotes the activation of protein C by thrombin, which can be referred to as protein C activation cofactor activity. It promotes the inactivation of thrombin by antithrombin, which can be referred to as antithrombin-dependent anticoagulant activity. Finally, it prevents the cleavage of fibrinogen by thrombin, which can be referred to as direct anticoagulant activity.
The complete TM molecule is quite anionic (pI.apprxeq.4), in part because of extensive post-translational glycation of the large extracellular domain of TM with an unusual hypersulfated, chondroitin E-like glycosaminoglycan (GAG) moiety. This bulky polyanionic domain strongly influences the three anticoagulant functions of TM. See, M. -C. Bourin et al., J. Biol. Chem., 265, 15424-15431 (1990), K. Nawa et al., Biochem. Biophys. Res. Commun., 171,729-737 (1990), K. T. Preissner et al., J. Biol. Chem., 265, 4915-4922 (1990), and J. F. Parkinson et al., Biochem. J., 283, 151-157 (1992).
The interaction of highly (pI&gt;11) cationic eosinophil-specific granule proteins with anionic TM has also been examined. It was believed that these toxic cationic proteins (major basic protein, eosinophil peroxidase, eosinophil cationic protein, and eosinophil-derived neurotoxin) might bind to TM through electrostatic interactions and impair its functions. See, A. Slungaard et al., J. Clin. Invest,, 91, 1721-1730 (1993). Several such proteins, including major basic protein (MBP), were found to potently inhibit (IC.sub.50 of 1-2 .mu.M) the capacity of endothelial cell TM to generate APC. MBP also inhibited APC generation by purified, soluble rabbit TM with an IC.sub.50 of 100 nM (roughly 1 .mu.g/ml). This inhibition was reversed by polyanions such as chondroitin sulfate E or heparin. The interaction between MBP and TM occurred with the large O-linked GAG moiety because a recombinant TM polypeptide comprising the soluble extracellular domain including the GAG was strongly inhibited by MBP whereas its counterpart lacking the GAG was not so inhibited. Thus, eosinophil cationic granule promins potently inhibited anticoagulant activities of the glycosylated (GAG+) form of TM, thereby suggesting a potential mechanism to explain the pronounced thromboembolic diathesis that characterizes hypereosinophilic conditions. Based on these experiments with the eosinophil cationic proteins, it has been predicted that Platelet Factor 4 and other cationic proteins might similarly inhibit TM function and thereby promote clotting in the presence of activated platelets.
The present invention is based on the discovery that Platelet Factor 4 and other relatively nontoxic cationic proteins do not function as do the highly toxic eosinophil cationic granule proteins. Thus, the present invention provides a composition containing cationic proteins that promotes the inhibition of blood coagulation. Such compositions find utility in a variety of clinical settings including treatment of myocardial infarction, pulmonary embolism, cerebrovascular disease and the like.